Prevention of epithelial cancer

ABSTRACT

The present invention relates to a method for prevention of epithelial cancer such as lung, bladder, prostate or gynaecological cancer or the initiation and/or progression of epithelial cancer such as lung, bladder, prostate or gynaecological cancer in a human comprising administering to the human of a combination dosage of a cyclooxygenase (COX) inhibitor, a vitamin D 3  including analogues and metabolites thereof and/or calcium. In a further aspect the invention relates to a method for reducing the effective dosage of ASA in a chemopreventive treatment of epithelial cancer in a human by co-administration with a non toxic dosage of a vitamin D 3  including analogues and metabolites thereof and/or Ca in the form of a combination dosage. In another embodiment the invention relates to the use of a cyclooxygenase (COX) inhibitor, a vitamin D 3  including analogues and metabolites thereof and/or calcium for the preparation of a medicament for the prevention of epithelial cancer such as lung, bladder, prostate or gynaecological cancer or the initiation and/or progression of epithelial cancer such as lung, bladder, prostate or gynaecological cancer.

[0001] In recent years, focus is very much on cancer prevention, inacknowledgement of the fact that surgery mostly does not suffice as theonly modality and that most cytotoxic regimens are ineffective againstsolid tumours. With the choices and results of treatment known today,only effective prevention will be able to reduce decisively epithelialcancer such as lung, bladder, prostate and gynecological cancermorbidity and mortality in a decisive manner.

[0002] The term chemoprevention covers the use of pharmacologicallyactive, non-cytotoxic agents or naturally occurring nutrients thatprotect against the emergence and development of clones of mutated,malignant cells.

[0003] The present invention relates to use of a cyclooxygenase (COX)inhibitor, vitamin D₃ including analogues and metabolites thereof orcalcium as well as combinations thereof for the preparation of amedicament for chemoprevention of epithelial cancer, such as lung,bladder, prostate and gynaecological carcinogenesis. In a further aspectthe invention relates to a method for reducing the effective dosage of acyclooxygenase (COX) inhibitor such as acetylsalicylic acid (ASA) in achemopreventive treatment of epithelial cancer, such as lung, bladder,prostate and/or gynecological cancer in a human by co-administrationwith a non toxic dosage of a vitamin D₃ including analogues andmetabolites thereof and, if so desired, Ca. In a presently preferredembodiment the invention relates to the use of ASA, a vitamin D₃ andcalcium together with a pharmaceutically acceptable carrier for thepreparation of a medicament for preventing the initiation and/orprogression of epithelial cancer, such as lung, bladder, prostate and/orgynaecological cancer in a human. The invention also relates to suchpharmaceutical medicaments.

BACKGROUND

[0004] Calcium

[0005] Calcium salts are used mainly in the treatment of calciumdeficiency. Hypocalcaemia, a decrease in plasma-calcium concentrationbelow the normal range, may be due to reduction in dietary intake,impaired or reduced absorption from the gastrointestinal tract,increased deposition in bone, or to excessive losses. Other causes ofhypocalcaemia include decreased parathyroid hormone activity, vitamin Ddeficiency, and hypomagnesaemia.

[0006] Pharmacokinetics and Dynamics

[0007] Calcium is absorbed from the small intestine and vitamin Dstimulates this absorption. About one-third of ingested calcium isabsorbed although this can vary depending on dietary factors and thestate of the small intestine; also, the absorption is increased duringperiods of high physiological requirement such as during pregnancy andlactation.

[0008] After absorption calcium is eventually incorporated into bonesand teeth with 99 percent of the body's calcium content being present insuch skeletal tissue. The remaining calcium is present in both theintra- and extracellular fluids. About 50 percent of the totalblood-calcium content is in the physiologically active ionised form with5 percent being complexed to citrate, phosphate, or other anions and 45percent being bound to proteins. The amount of calcium in the intestinallumen binds free fatty acids and secondary bile acids by saponificationand reduces the local irritant effect of these acids in the colon.

[0009] Excretion of calcium occurs in the urine although a largeproportion is reabsorbed in the renal tubules. Excretion also occurs inthe faeces, this consisting of unabsorbed calcium as well as thatsecreted in the bile and pancreatic juice. Minor amounts are lost in thesweat. Calcium crosses the placenta and is also excreted in breast milk.

[0010] Mechanisms of Action

[0011] In recent years focus has Increasingly been directed at thecentral role of calcium in intra- and intercellular signal transduction.

[0012] Calcium is a key factor in maintaining normal cell membranefunction, and calcium flux over the cell membrane plays a central rolein mediating intracellular signal transduction regulating multiplecellular functions. Furthermore, the expression of cellular surfacecadherins, which are necessary to maintain Intercellular contact,depends on the presence of calcium. Thus, calcium apparently has animportant role in the structure and dynamics of the cellularactin-cytoskeleton.

[0013] Reduction of calcium concentration in intercellular fluid lowerscell response to growth regulating factors and reduces the permeabilityof cell membranes. When the calcium concentration is reduced, the ratesof proliferation and dedifferentiation increase. Proliferative signalsfrom low extracellular calcium are emitted via increased expression ofc-myc.

[0014] Calcium contributes to the regulation of all cell division andcell differentiation phases, primarily through activation of variousprotein kinases (cAMP dependent kinase, Ca-calmodulin dependent proteinkinase, protein kinase C).

[0015] Administration

[0016] The recommended daily allowance (RDA) of calcium is 800 to 1200mg daily, increasing to 1500 mg daily for the elderly.

[0017] Recommended dosage of calcium depends on the patient's disorder.In dietary deficiency and as adjunct to osteoporosis therapy doses of1000 to 1500 mg daily are recommended. Osteomalacia is usually treatedwith 1000 to 3000 mg of calcium.

[0018] 1,25-dihydroxycholecalciferol (Calcitriol)

[0019] Vitamin D compounds are fat-soluble sterols, whose activemetabolites are considered to be hormones, which are essential forproper regulation of calcium and phosphate homeostasis and bonemineralisation.

[0020] Toxicity

[0021] Excessive intake of vitamin D leads to the development ofhypercalcaemia and its associated symptoms including anorexia, nausea,vomiting, weakness, fatigue, and headache. Interindividual tolerance tovitamin D varies considerably; infants and children are generally moresusceptible to its toxic effects. Milk-alkali syndrome withhypercalcaemia is a rare possibility in chronically treated individuals.

[0022] Certain synthetic vitamin D₃ analogue preparations have proved tohave 100 to 200 times the anti-proliferative effect and effect ondifferentiation and only 0.5 times the hypercalcaemic effect of1,25-dihydroxycholecalciferol (H. H. Raskov, 1999, not published, Zhouet al., 1991 and Evans et al., 2000).

[0023] Pharmacokinetics and Dynamics

[0024] Vitamin D substances are well absorbed from the gastrointestinaltract. The presence of bile is essential for adequate intestinalabsorption; absorption may be decreased in patients with decreased fatabsorption.

[0025] Vitamin D and its metabolites circulate in the blood bound to aspecific α-globulin. Vitamin D can be stored in adipose and muscletissue for long periods of time. It is slowly released from such storagesites and from the skin where it is formed in the presence of sunlightor ultraviolet light. Vitamin D compounds generally have a slow onsetand a long duration of action; the newer analogues and metabolites,however, have a more rapid action and shorter half-lives.

[0026] Cholecalciferol (vitamin D₃) is hydroxylated in the liver to form25-hydroxycholecaliferol D(25(OH)D₃). This compound undergoes furtherhydroxylation in the kidneys to form the active metabolite1,25-dihydroxycholecalciferol (1α,25(OH)₂D₃). Further metabolism alsooccurs in the kidneys.

[0027] Vitamin D compounds and their metabolites are excreted mainly inthe bile and faeces with only small amounts appearing in the urine;there is some enterohepatic recycling but it is considered to have anegligible contribution to vitamin D status. Certain vitamin Dsubstances may be excreted into breast milk.

[0028] Vitamin D increases serum calcium by facilitating the absorptionof calcium and phosphate from the intestinal canal as well as mobilisingcalcium from bones. It is known from studies of bone mineral turnoverthat vitamin D and calcium are mutually dependent factors, and this hasalso proved to be the case in the regulation of cell division and celldifferentiation.

[0029] Mechanisms of Action

[0030] In addition to similarities in structure, metabolism and actionto steroid hormones, vitamin D also crosses the cellular plasma membraneby diffusion and binds to specific cytoplasmic receptors (vitamin Dreceptor, VDR). Upon binding and activation this hormone-receptorcomplex is transported into the cell nucleus, where it activatestranscription and subsequent protein synthesis.

[0031] A vitamin D receptor specific for the active metabolite ofvitamin D₃ 1,25-dihydroxycholecalciferol has been found in the cytoplasmof a wide variety of tissue and cells. After binding to the receptor,the ligand-receptor complex is translocated into the nucleus where itcomplexes with the RXR (Retinoic X Receptor). This VDR-RXR hetereomerbinds to promoter regions of cell cycle regulating genes containing avitamin D response element.

[0032] The cyclin-dependent kinase inhibitor (Cki) p21^(Waf-1/Clp-1) isone of the genes regulated in this way. It regulates the cell cycle byinhibition of the cyclin dependent kinases (Cdk) and thereby preventingcells passing from the G1 to the S phase. 1,25-dihydroxycholecaliferolalso modulates c-myc, c-fos and c-jun oncogenic expression andpresumably induces apoptosis.

[0033] Besides 1,25-dihydroxycholecalciferol, proliferation anddifferentiation is also regulated by the growth factors transforminggrowth factor, TGF and epidermal growth factor, EGF via SMAD signalproteins and VDR. This allows for exchange of signals between vitamin Dand TGF and EGF signalling pathways (cross talk). At increased EFGRactivation VDR is down-regulated.

[0034] 1,25-dihydroxycholecalciferol increases intracellular calcium andstimulates various protein kinases by increased tyrosine phosphorylationand activation of various signal transmission systems.1,25-dihydroxycholecalciferol stimulates transcription of the calbindinD gene in colonocytes, which is believed to increase transcellularcalcium absorption.

[0035] Administration

[0036] A dose of 10 μg (400 IU) cholecalciferol (vitamin D₃) is usuallysufficient in adults for the prevention of simple deficiency states.Deficiency due to malabsorption states or liver disease often requireshigher doses for treatment of up to 1 mg (40,000 IU) daily.

[0037] The 1,25-dihydroxycholecalciferol (calcitriol) metabolite ofcholecalciferol is often given as an initial adult dose of 0.25 to 2 μgdaily, increased if necessary to a maximum of 3 μg daily.

[0038] Cyclooxygenases (COX)

[0039] The two identified isomeric forms of the COX enzymes, COX-1 andCOX-2, catalyse the rate-limiting step in the synthesis of prostaglandinand therefore also known as prostaglandin synthases. The COX-1 isexpressed as a constitutive active enzyme in virtually all tissues. Inthe upper gastrointestinal tract it affects the mucosa barrier byinducing bicarbonate secretion and mucin production primarily throughprostaglandin E (PGE). PGE is quantitatively the dominant product of theturnover of arachidonic acid induced by COX-1.

[0040] Apart from the brain and kidneys where COX-2 is constitutiveactive, COX-2 is an inducible enzyme. Particularly, COX-2 is induced byinflammatory stimuli, it catalyses the formation of pro-inflammatorycytokines, including PGE₂ and PGF_(α) which induce proliferation,suppress the immune system and stimulate angiogenesis.

[0041] The substrate for the COX-2 enzyme is arachidonic acid which ismetabolised via three different pathways (see FIG. 1): via the COXpathway to eicosanoids, which stimulate cell division, as observed ininflammatory conditions, or via the lipoxygenase pathway tohydroperoxides (HPETE) and hydroxy compounds (HETE). The third pathwayfor arachidonic add metabolism is via cytochrome P450 to HETE and EET(epoxyeicosatrienic acid).

[0042] Several classic carcinogens are used as electron donors duringthe COX catalysed reaction and are thereby activated (these agents havehigh DNA affinity). Among these carcinogens are polycyclic aromatichydrocarbons, aflatoxins, halogenated pesticides, aromatic amines andphenol compounds. Thus, COX activates potential carcinogens into activemetabolites harmful to the DNA.

[0043] The exact biochemical and cellular mechanisms underlying thepreventive effect of COX inhibitors is only partially understood, but isconsidered to be closely related to the impact these drugs have onarachidonic acid metabolism and prostaglandin synthesis.

[0044] Mechanisms of Action

[0045] The COX inhibitor acetylsalicylic acid (ASA) (e.g. Aspirin) andits metabolite salicylate block the formation of prostaglandin fromarachidonic acid by irreversible acetylation of COX, thereby preventingarachidonic acid access to the active part of the enzyme. The COXactivity can only be re-established through production of new COXmolecules, and cells without protein synthesis, such as platelets, aretherefore unable to regain COX activity. The predominant chemopreventiveeffect of ASA is deemed to be COX-2 inhibition, which results inmetabolism of arachidonic acid via a lipoxygenase pathway to 15-HETE(leucotriene with anti-inflammatory and antimitogenic effects), seeFIG. 1. Most other NSAIDs (e.g. piroxicam, sulindac and indomethacin)exhibit reversible and dose-dependent blockage of COX. This makes ASA amore potent inhibitor of prostaglandins. As it appears from the above,however, there are several mechanisms of action. The prostaglandincascade also depends on the calcium-regulated signal transmissionsystem.

[0046] NSAID has been shown to inhibit several endonucleases; these areenzymes that cleave DNA molecules. Presumably they play a central partin the genomic instability that is one of the characteristics ofepithelial multistep carcinogenesis (Hanif et al., 1995). Othermolecular biology mechanisms are discussed in detail in (Kahlenberg etal., 1998).

[0047] Other proneoplastic effects of COX include changing TGF_(β) froman anti-proliferative into a pro-proliferative growth factor, reducingintercellular and cellular-stromal contact and communication, andstimulating of angiogenesis and metastasis (Hanif et al., 1995).

[0048] The COX-1 protein contain three domains (COX domain, EGF likedomain and membrane binding motif), of which one resembles the EGF (andalso TGF_(α), which belongs to the EGF family). Inhibition of COX-1 isalso associated with anti-angiogenesis, although to a lesser extent thanCOX-2 inhibition (DuBois, RN, personal communication).

[0049] The above mentioned properties of the COX isoforms seem toindicate that inhibition of both isoforms (COX-1 and COX-2) may be ofimportance to achieve optimum prevention.

[0050] The most frequent undesirable effects connected with long-termadministration of NSAIDs are gastroduodenal ulceration and bleedingbecause of low PG and thromboxane A₂ levels in the gastrointestinaltract. PG stimulates mucin production and bicarbonate secretion, andthromboxane A₂ indicates platelet aggregation. These complications areprimarily related to inhibition of the constitutive COX₁ enzyme.

[0051] Undesirable effects and complications primarily relate to the useof NSAIDs as analgesic or anti-inflammatory agents in significantlyhigher doses, but they are potential sequels after long-term use also inlower doses.

[0052] A review of 16 cohort studies and case-control studies showedthat the risk of developing severe NSAID-induced gastrointestinalundesirable effects amounts to 2 to 4 percent a year at analgesic andanti-inflammatory daily doses. In low-dose aspirin prophylaxis ofcardiovascular disease the relative risk-reduction in relation tostroke, acute myocardial infarction and/or cardiovascular death wasfound to be approx. 25%.

[0053] The Physicians' Health Study (325 mg of acetylsalicylic acid qod)found that, in addition to a significant reduction of the risk of acutemyocardial infarct, there were significantly more cases of melaena andepistaxis than in the placebo group, but of neither cerebral haemorrhagenor unspecific gastrointestinal bleeding (including haematemesis).

[0054] There exist numerous data on the pharmacokinetics and toxicity ofCOX inhibitors, especially regarding ASA. The FDA has found that forinstance acetylsalicylic acid is a safe and efficient anti-inflammatoryand analgesic agent and well suited for over-the-counter sales. Nofurther toxicological studies are necessary to assess the usage ofacetylsalicylic acid in chemoprevention.

[0055] Lung Cancer

[0056] Lung cancer is one of the most prevalent and lethal cancers(USA>160,000 deaths, EU>150,000). More than 90 percent are caused byexposure to tobacco smoke (on virtually all instances cigarettesmoking). There are four distinct histological types: Small cellcarcinoma 20% Adenocarcinoma 35% Non Small Cell Lung Cancer Squamouscell carcinoma 30% Non Small Cell Lung Cancer Large cell carcinoma 15%Non Small Cell Lung Cancer

[0057] 5-year survival for NSCLC is 10%, for SCLC 3%. Surgery andchemotherapy have virtually no impact on the mortality. Only primaryprevention will have an impact on survival.

[0058] Animal Models

[0059] Animal models of lung cancer are using either chemically inducedcarcinogenesis (i.e. nitrosamines, benzopyrenes) or geneticallyengineered models (i.e. p53 transgenic rodents). The chemically inducedcarcinogenic models are excellent study systems as they mimic thegenetics and molecular biology of human lung cancer (Kang Y et al.,2000). One of the characteristics of human lung cancer development isthe histological adenoma-carcinoma sequence from normal epitheliathrough a typical hyperplasia, early adenoma, late adenoma to invasivecarcinoma. Many of the mutations in gene and down-stream effects areidentical to other forms of solid cancers, i.e. mutations In K-ras, APC,MCC, p53 also seen in epithelial cancer. Essential signalling pathwaysalso identical in these types of cancer, i.e. the wnt-, and EGFRpathways (Sunuga N et al., 2001, Tsujiuchi, T et al., 2000).

[0060] COX Expression in Lung Cancer

[0061] Increased COX-2 expression has been found in human lung cancer(Soslow R A et al., 2000). The inhibition of COX in animal settings isassociated with conflicting results: COX inhibition (ASA) in a mousemodel resulted in an increase in apoptotic index but no impact on numberof K-ras mutations (Yao R. et al, 2000).

[0062] COX inhibition (ASA) in a mouse model (A/J) showed no effect(Witschi H., 2000).

[0063] In another study of COX2 inhibition in A/J mouse model ASA andNS-398 treatment decreased the mean of the lung tumor volumes (Rioux Net al., 1998). A study comparing the chemopreventive efficacies ofaspirin and sulindac against tumorgigenesis in A/J mice concluded thatsalicylate and sulindac could be equally effective as chemopreventiveagents (Duperron, C. et al., 1997)

[0064] So far only 5 chemopreventive studies in human populations havebeen identified by the IARC (International Agency for Research ofCancer, Lyon, France):

[0065] 1 beta-carotene+retinal

[0066] 2 Retinol

[0067] 3 COX2 Inhibition (Celebra, Pharmacia)

[0068] 4 Upoxygenase inhibition (Zileutenin)

[0069] 5 N-acetylcysteine

[0070] The study on COX2-inhibition is entitled Biological Effect andTolerability of Celecoxib as a Chemopreventive Agent in Current andFormer Smokers by Hong, W. K is a randomised placebo-controlledbiomarker-based clinical trial in current and former smokers examiningthe effect of the COX-2 inhibitor celecoxib on biomarkers of lung cancerrisk and COX-2 dependent signaling pathways. (Hong, W. K.)

[0071] None of the above studies have been terminated (termination2002-2005) and there is so far no indication of effectivechemoprevention against lung cancer.

[0072] Bladder Cancer

[0073] The annual incidence is about {fraction (23/100,000)} inhabitants({fraction (36/100,000)} men and {fraction (10/100,000)} women).Five-year survival rate is 40-66 percent. 40% of the patients with CISdevelop invasive cancer within 5 years. Tobacco (primarily cigarette)smoking accounts for 30-40 percent of all bladder cancers. Other riskfactors are occupational exposure to rubber, organic dye and metalrefining, cyclophosphamide, schistosomal infection and geneticpredisposition ex. HNPCC (2%) Frequently involved genes/mutations/growthfactors are p16, p53, c-myc, k-ras, EGF, Rb., COX2 (Cordon-Carlo, C. etal. 2000, Gonzalgo, M L et al. 2000, Wijkström, H. et al., 2000,Kömhoff, M. et al., 2000).

[0074] More than ninety percent are transitional cell carcinomas, 8percent squamous cell carcinomas and 2 percent adenocarcinomas.Papillary urothelial hyperplasia is an early intermediate biomarker fortransitional cell bladder cancer

[0075] Present treatment of Ta and T1 tumours is TURB. Tumours are lowgrade and superficial. 50-70 percent recur locally within 6-12 months.For Tcis treatment is intravesical BCG installation or surgery(resection/cystectomy), whereas for T2-T4 tumours the treatment isresection/cystectomy.

[0076] Animal Models

[0077] The most frequent animal models are the carcinogen stimulatedrodent models using either male B6D2F1 mice or female F344 Fischer rats.

[0078] Literature Search

[0079] A search in the National Library of Medicine using the PubMed on

[0080] bladder neoplasia (BN) and chemoprevention (CP) revealed anepidemiological study on NSAIDS, primarily on Phenacetin (OR=0.81)(Castelao et al., 2000). According to IARC no ongoing chemopreventivestudies in human bladder cancer populations are registered.

[0081] Searching the National Library of Medicine using the PubMed onBladder neoplasms and aspirin a small body of data revealing conflictingevidence is found. In the literature aspirin is referred to as both anactive chemopreventive agent (Klan R. et al., 1993), as an inactiveagent (Rao et al., 1996), and even as an active promotor forcarcinogenesis (Cohen et al., 1989).

[0082] When looking at bladder neoplasms and cholecalciferol onecell-line study on 253j and T-24 cell lines results in significantreduction in proliferation and induction of apoptosis (Konety et al.,2001).

[0083] Focusing on existing literature on bladder neoplasms it allconcentrate on bone metabolism, hypercalcaemia, bone metastasis with noreference to epidemiology and prevention.

[0084] In conclusion there are no data in the existing literature toactive chemoprevention of bladder neoplasm with reference to acombination of a COX inhibitor, cholecalciferol or its metabolites orcalcium.

BRIEF DESCRIPTION OF THE INVENTION

[0085] No prospective, randomised, double-blind studies on prevention ofepithelial cancer such as lung, bladder, prostate and gynecologicalcancer based upon a cyclooxygenase (COX) inhibitor, vitamin D₃ includinganalogues and metabolites thereof or calcium as well as combinationsthereof exist. Studies of cancer chemoprevention are extremelyexpensive, as these would require a very high number of individuals andmany years of duration if the study endpoints were to be invasive cancerand cancer-related mortality. For these reasons there is an increasingtendency towards relying on epidemiological studies of intermediaryendpoints, animal trials of genetically engineered or carcinogenicallystimulated animal populations and biological models for examiningdifferent biomarkers (mutations, growth factors, etc).

[0086] The individual drugs exert their effects on specific areas of thecarcinogenesis: modification of signal transduction and expression ofoncogenes, reduction of the carcinogenic impact on colonic epitheliumand intracellular and intercellular signal transduction, inhibition ofCOX enzymes and probably apoptosis.

[0087] Based on epidemiological, animal experimental, and molecularbiology studies, it is hypothetically possible to achieve an additive orsynergistic cancer-preventive effect of a combination of the threesubstances described herein.

[0088] By combining ASA, 1,25-dihydroxycholecalciferol (or an analoguevitamin D preparation) and calcium, an additive or synergistic effect islikely to be achieved. Therefore, concentrations of the individualpreparations can be decreased and toxicity reduced to a negligiblelevel. Furthermore, compliance is more easily achieved with a singlepreparation than with a combination of three different products.

[0089] According to the present invention, the epithelial cancer, suchas the lung, bladder, prostate and gynecological cancer preventiveeffect of the preparations manifests itself by a significant reductionof the incidence and overall morbidity and mortality of epithelialcancer, such as lung, bladder, prostate or gynaecological cancer. Toachieve this effect, however, it is believed to be important to take thepreparation consistently as prophylaxis over a long time (probably morethan one year), exactly as for the prevention of ischaemic heart diseaseand osteoporosis.

[0090] By combining ASA with Calcitriol 1,25DHC (or an analoguepreparation) or with calcium, an additive or synergistic effect isachieved, so that the amounts of the individual drugs presumably arereduced and the toxicity thereby reduced to a negligible level. In apreferred embodiment, ASA is combined with both 1,25DHC (or an analoguepreparation) and calcium.

[0091] The preparations could be combined as follows: 500 mg of Calcium(calcium carbonate 1250 mg) and/or 0.5 μg of 1,25DHC (or vitamin D₃ 400IU or D₃ analogue, eg, 0.25 μg of calcitriol or 0.005 μg ofcalcitriol/kg BW) and 75 mg of ASA or an analogues reversible orirreversible COX₂ Inhibitor

[0092] The main requirements for a preparation designed forchemoprophylaxis include: low price, high compliance and ultra-lowtoxicity; it is assumed that by adding 1,25DHC and calcium the amount ofCOX inhibitor (ASA) can be reduced, so that the ASA-related undesirableeffects can be reduced to a negligible level without reducing itsaction.

[0093] At first, in vivo studies of the effect of the above have beencarried out in the form of animal experiments in collaboration withPipeline Biotech A/S. The results of the studies are shown in Table 1.

[0094] As a chemopreventive effect has been demonstrated on epithelialcancer as evidenced by the results shown in the example of the presentapplication demonstrating a highly significant effect of a combinationof a cyclooxygenase inhibititor, vitamin D3 or an analogue or metabolitethereof and calcium on lung and bladder carcinogenesis, chemopreventionfrom the use of can also be expected on other epithelial cancers such asprostate cancer and gynaecological cancer carcinogenesis. By the termgynaecological cancer is meant a cancer selected from the groupconsisting of ovarian epithelial cancer, such as serous cystomas,mucinous cystomas, endometrioid tumors, clear cell tumors andunclassified tumors of the ovarie; endometrial cancer, such as ciliatedadenocarcinoma, secretory adenocarcinoma, papillary or villoglandular,adenocarcinoma with squamous differentiation, adenoacanthoma,adenosquamous; and cervical cancer such as squamous cell (epidermoidcarcinoma) and adenocarcinoma.

[0095] In human and in animal studies, the first steps in the multistepcarcinogenesis characterizing cancer development in lung mucosa, urinarybladder mucosa and mucosal tissues of the reproductive system have manyidentical aspects to epithelial cancer with regard to pathology,involved genetic changes (mutations) and down-stream effects hereofirrespective in which of the above organs they arise.

[0096] Mutual events in molecular biology during carcinogenesis in therespiratory tract, gastrointestinal tract, urinary tract andreproductive system are seen in a range of different cellularmechanisms:

[0097] Genetic changes during early carcinogenesis: APC mutations andbeta-catenin mutations (Tsujichi, T. et al., 2000, Sunage, N. et al.,2001), DNA hypomethylation (Piyathflake, C J. et al., 2000), p53mutations (during promotion/progression)

[0098] Down-stream effects on growth-promoting oncogenes and growthfactors: myc and ras upregulation (Hirsch, F R. et al., 2001), EGFoverexpression (Saha, D. et al., 1999)

[0099] Down-stream effects on growth-inhibiting tumour suppresor genesand growth factors: reduced levels of expression of TGF-beta (Kim, T. etal., 2001)

[0100] Down-stream effects on enzyme expression and metabolism: COX2overexpression (Marrogi, A). et al., 2000, Hosomi, Y. et al., 2000,Shirharama, T., 2000, Ristimaki, A. et al., 2001), COX1 overexpression(Rioux, N. et al., 2000), increased PgE(2) synthesis Saha, D. et al.,1999, Rioux, N. et al., 2000)

[0101] In animal studies on lung cancer, non-steroidal anti-inflammatoryagents (NSAID) are shown to counteract the carcinogenesis initiated bythe lung-tumour producing chemicals (Witschi, H. et al., 2000).

[0102] Accordingly, the scope of the present invention encompassesmethods of treatments directed towards chemoprevention for lung cancercarcinogenesis, urinary bladder carcinogenesis, prostate carcinogenesisand gynaecological cancer carcinogenesis, and pharmaceuticalcompositions as outlined above for the use therefor. To investigate thechemopreventive effect of the combination of a cyclooxygenase inhibitor,1,25 Dihydroxycholecaliferol and calcium, research protocols weredeveloped to conduct animal experimental trials using appropriate rodentmodels of chemically induced carcinogenesis well known to the person ofskill in the art. The experimental rodent models are attractive becausea substantial overlap exists between man and mouse/rat in the geneticalterations thought to be responsible for tumourigene-sis. An example ofsuch a trial is a trial using the A/J mouse lung cancer model whereinthe carcinogen stimulation is provided by a benzpyrene product deliveredby a gastric tube with various combinations of ASA, 1,25 DHC andcalcium. The intermediary endpoints are precursor lesions such as atypical adenomatous hyperplasia of the lung or urothelial dysplasia,papillary hyperplasia and/or papilloma.

DETAILED DESCRIPTION OF THE INVENTION

[0103] In one embodiment the invention relates to a method for reducingthe effective dosage of ASA in a chemopreventive treatment of epithelialcancer, such as lung, bladder, prostate and gynaecological cancer in ahuman at risk by co-administration with a non toxic dosage of acalcitriol including analogues and metabolites thereof and/or Ca in theform of a combination dosage. This is due to the fact that it hassurprisingly been shown that the addition of the calcitriol and calciumby synergy in action decrease the necessary dosage of acetylsalisylicacid in order to decrease the formation of abberant crypt foci in a ratmodel of epithelial cancer both with respect to size and numbers (seeWO01/22974). In a presently preferred embodiment, the invention relatesto a method for prevention of the initiation and/or progression ofepithelial cancer, such as lung, bladder, prostate and gynaecologicalcancer in a human comprising administering to the human a combinationdosage of a cyclooxygenase (COX) inhibitor, a calcitriol includinganalogues and metabolites thereof and calcium.

[0104] By chemopreventive effect is meant prevention of epithelialcancer, such as lung, bladder, prostate and gynaecological cancer or theinitiation and/or progression of epithelial cancer, such as lung,bladder, prostate and gynaecological cancer and or the effect ofreducing the formation of conditions being pre-malignant of lung,bladder, prostate or gynaecological cancer.

[0105] By a combination dosage according to the present invention ismeant individual dosages for instance in one packet containing therelevant pharmaceuticals in appropriate dosages and accompanied byinstructions for a treatment regimen according to the use and methods ofthe invention or a physical entity of two or more of the ingredients.The combination dosage may also comprise different COX inhibitors aswell as different vitamin D₃ analogues and/or metabolites.

[0106] According to the present invention, it is believed that theadministration of the combination dosage should be carried out regularlywith an average daily dosage of the cyclooxygenase inhibitorcorresponding to the inhibition of COX₁ and/or COX₂ provided by a dosageof ASA in the range of 50 mg to 500 mg, preferably in the range of 25 to400 mg, more preferred of in the range of 50 to 300 mg, still morepreferred in the range of 75 to 150 mg, such as in the range of 75 to100 mg, e.g. measured by whole blood assays e.g. by the method disclosedin Ehrich, E. et al., 1999.

[0107] The combination dosage may further comprise a vitamin D₃including analogues and metabolites thereof corresponding to theantiproliferative and/or cell differentiation effect of the vitamin D₃metabolite 1,25 dihydroxycholecalcipherol in the range of 0.1 microgramto 2 microgram.

[0108] Any calcium in the combination dosage may preferably be in therange of 200 mg to 3500 mg such as calcium in the range of from 250 mgto 3000 mg, such as in the range of from 300 mg to 2500 mg, preferablyin the range of 400 to 2000 mg, more preferred in the range of from 500to 1000 mg, such as 750 mg.

[0109] As mentioned above, the treatment or prevention should becontinued for a long period in order to give the best effect; however itis believed that a beneficial effect may be obtained after a treatmentof at least 3 months. Accordingly, the administration is preferablycontinued for at least 6 months, such as at least for 1 year, preferablyfor at least 2 years. However, persons in high risk may be treatedaccording to the present invention for the rest of their lives.

[0110] In an important aspect, the administration of the combinationdosage results in the prevention of the initiation or progression ofepithelial cancer exceeding the effect of the administration of any ofthe individual ingredients in the same daily dosage and in the sameperiod.

[0111] In another aspect, the method according to the invention is onewherein the combination dosage has a preventive effect which is at leastadditive compared with the effect of the individual effectiveingredients. In one embodiment, the preventive effect is at leastsynergistic compared with the effect of the individual effectiveingredients.

[0112] An additive effect according to the invention may be calculatedas an effect of the sum of prevention by each of the substances ASA,Vitamin D₃. and Ca, respectively or by an effect of the sum ofprevention by the selection of two of the substances ASA, the VitaminD₃, and the Ca and the preventive effect by the remaining substance.

[0113] By synergistic effect according to the present invention ispreferably meant an effect which is higher than the additive effect asdisclosed above.

[0114] The COX inhibitor may be any one acting on one or more of themechanisms selected from non-selective, reversible or irreversibleacetylation of COX, reversible or irreversible acetylation of COX₂,reversible or irreversible acetylation of COX₂, inhibition ofangiogenesis, inhibition of arachidonic acid metabolism, blocking of AAmetabolism, inhibition of the stimulation of proliferation stimulationfrom Epidermal Growth Factor, and stimulation of apoptosis. In apreferred embodiment, the COX inhibitor is primarily a COX₂ inhibitor.The preferred COX inhibitor is ASA.

[0115] The ASA is preferably acting by one or more of the followingmechanisms: inhibiting of cell proliferation; inhibition of upregulationof pro-proliferative agents such as growth factors; modulation of signaltransduction; and induction of apoptosis. Also inhibition ofangiogenesis, and inhibition of arachidonic acid metabolism may be thetarget for the ASA. In a further aspect, the cyclooxygenase inhibitoracts by decreasing the formation of potential carcinogens into DNAharmful metabolites.

[0116] The vitamin D₃ including analogues and metabolites thereof arepreferably acting by one or more of the following mechanisms: inhibitionof cell proliferation; inhibition of DNA synthesis; modulation of signaltransduction; induction of differentation; and induction of apoptosis.

[0117] In an important aspect of the invention, the vitamin D₃ is asynthetic analogue having an hypercalcaemiac effect of 0.5 of thehypercalcaemiac effect of 1,25 dihydroxycholecalcipherol. A number ofsynthetic vitamin D analogues suitable according to the presentinvention are disclosed herein. In order to substitute one vitamin D₃analogue according to the present invention, the relevant dosage may becorrelated to the effect of 1,25 dihydroxycholecalcipherol by referenceto the antiproliferative effect which may be evaluated by methodswell-known in the art, such as disclosed in Wargovich, M J. et al., 1987and Eisnan, J A. et al., 1987 herein. Alternatively, PTH may be used forthe comparison study, e.g. measured as disclosed In Johansson S. et al.;2001.

[0118] According to the present invention, the active mechanism of thecalcium is preferably an effect on the expression of cellular surfacecadherins and/or intra- and extracellular signal transmission.

[0119] One very important aspect of the present invention is the findingthat the method may reduce the risk of developing epithelial cancer,such as lung, bladder, prostate or gynaecological cancer in theindividual human receiving the treatment by at least 10% or morecompared to the effect obtained by any of the individual ingredients inthe same dosage and in the same period of administration. The reductionmay be at least 20% or more, and in certain circumstances e.g. for highrisk patients even 30% or more, preferably about 50%. The period usedfor measurement may be at least 3 months, such as at least 6 months,most preferred at least 1 year, such as at least 2 years.

[0120] One preferred combination dosage according to the invention isthe combination dosage comprising ASA, 1,25 DHC and Ca in that theseIngredients are all well known drugs.

[0121] In a still further embodiment, the invention relates to the useof a vitamin D₃ and/or calcium together with a pharmaceuticallyacceptable carrier for the preparation of a medicament for preventingthe initiation and/or progression of epithelial cancer in a human. In apreferred embodiment the medicament is in the form of a combinationdosage comprising the vitamin D₃ and/or the calcium.

[0122] The use may be in accordance with any of the methods describedabove and in a still further embodiment, the invention relates to anysuch pharmaceutical medicament. The pharmaceutical medicament mayaccordingly comprise a combination of a cyclooxygenase (COX) inhibitor,a vitamin D₃ including analogues and metabolites thereof and/or calcium.In a further aspect, the pharmaceutical medicament is such a medicamentin any of the disclosed combination dosages which is to be administeredaccording to any of the methods described herein.

[0123] Accordingly, a further aspect of the invention relates to the useof a cyclooxygenase (COX) inhibitor, a vitamin D₃ including analoguesand metabolites thereof and calcium together with a pharmaceuticallyacceptable carrier for the preparation of a medicament for preventingthe initiation and/or progression of epithelial cancer in a human.

[0124] In a still further aspect, the present invention relates to apharmaceutical medicament comprising a combination of a cyclooxygenase(COX) inhibitor, a vitamin D₃ including analogues and metabolitesthereof and calcium in a combination dosage together with apharmaceutically acceptable carrier.

[0125] The pharmaceutical medicament according to the invention ispreferably a medicament wherein the combination dosage comprises vitaminD₃ including analogues and metabolites thereof corresponding to theantiproliferative and/or cell differentiation effect of the vitamin D₃metabolite 1,25 dihydroxycholecalipherol in the range of 0.1 μg to 2 μgsuch as in the range of 0.2μ to 1.5 μg, preferably in the range of from0.3 to 1 μg, more preferred in the range of from 0.4 μg to 0.75 μg, suchas 0.5 μg.

[0126] In a further embodiment, the pharmaceutical medicament is onewherein the combination dosage comprises calcium in the range of 200 mgto 3000 mg, such as In the range of from 300 mg to 2500 mg, preferablyin the range of 400 to 2000 mg, more preferred In the range of from 500to 1000 mg, such as 750 mg.

[0127] In a preferred embodiment the pharmaceutical medicament accordingto the invention is one wherein the combination dosage comprises ASA inthe range of 50 mg to 500 mg, preferably in the range of 25 to 400 mg,more preferred of in the range of 50 to 300 mg, still more preferred inthe range of 75 to 150 mg, such as in the range of 75 to 100 mg, Themost preferred medicament comprises 50 to 75 mg ASA, 500-1000 mg Ca, and0.5 to 1 μg of 1,25 dihydroxycholecalcipherol.

[0128] Accordingly, in a preferred embodiment, the pharmaceutical is onepharmaceutical comprising all three ingredients in order to secure theright individual dosage and patient compliance.

[0129] In a preferred embodiment, the pharmaceutical is a fixed-dosepharmaceutical comprising all three ingredients in order to secure thecorrect dosage. The pharmaceutical should be administered once or twiceper day.

[0130] In a preferred embodiment the D₃ vitamin has a limited Cainducing effect on the Ca level. Accordingly, preferably the D₃ is asynthetic analogue having a hypercalcaemic effect of the most 0.5 of thehypercalcaemic effect of 1,25 dihydroxycholecalcipherol, calculated on amolar basis. This may be measured by methods known in the art. In thefollowing relevant derivatives are disclosed.

[0131] In a still preferred embodiment, the vitamin D₃ analogues orderivatives are any one of the following disclosed in the references asmentioned.

[0132] WO 89/10351, which is hereby incorporated by reference, mentionsthe following vitamin D analogues:

[0133] A compound of the formula I

[0134] in which formula n is an integer from 1-7; and R¹ and R², whichmay be the same or different, represent for hydrogen, or straight orbranched, saturated or unsaturated C₁-C₇-alkyl; with the provisos thatwhen n=1, R¹ and R² cannot simultaneously be hydrogen, nor can R¹ and R²simultaneously be an alkyl group independently chosen from methyl, ethyland normal-propyl, and when n=2, R¹ and R² cannot simultaneously bemethyl; or C₃-C₈-cyclo-alkyl, or, taken together with the carbon(starred in formula I) bearing the hydroxyl group, R¹ and R² can form asaturated or unsaturated C₃-C₈ carbocyclic ring; and

[0135] R³ and R⁴ represent either both hydrogen, or when taken togetherconstitute a bond, such double bond (either in the Z or E configuration)connecting carbons numbered 22 and 23; and derivatives of the compoundsof formula I In which one or more hydroxy groups have been transformedinto —O-acyl or —O-glycosyl or phosphate ester groups, such maskedgroups being hydrolyzable in vivo, or derivatives of the compounds offormula I In which the hydroxyl group at the starred carbon atom islacking, these compounds being converted to active compounds of formulaI by enzymatic hydroxylation after administration.

[0136] In particular a disatereoisomer of a compound mentioned above, inpure form; or a mixture of diastereoisomers of a compound mentionedabove.

[0137] Moreover, a compound according to the above mentionedspecifications, selected from the group consisting of

[0138]1(S),3(R)-Dihydroxy-20(R)-(5-ethyl-5-hydrocy-1-heptyl)-9,10-secopregna-5(Z),7(E),10(19)-triene;

[0139]1(S),3(R-Dihydroxy-20(R)-(6-hydrocy-6-metyl-1-heptyl)-9,10-secopregna-5(Z),7(E),10(19)-triene;

[0140]1(S),3(R)-Dihydroxy-20(R)-(6-hydrocy-6-metylhept-1(E)-en-1-yl-9,10)-secopregna-5(Z),7(E),10(19)-triene;

[0141]1(S),3(R)-Dihydroxy-20(R-(6-ethyl-hydroxy-1-octyl)-9,10)secopregna-5(Z),7(E),10(19)-triene;

[0142]1(S),3(R)-Dihydroxy-20(R)-(7-hydrocy-7-metyl-1-octyl)-9,10)-secopregna-5(z),7(E),10(19)-triene;

[0143]1(S),3(R)-Dihydroxy-20(R)-(6′-metyl-1′-heptyl)-9,10-secopregna-5(Z),7(E),10(19)-triene;

[0144] WO 93/19044, which is hereby incorporated by reference, mentionsthe following vitamen D analogues:

[0145] A compound of the formula I

[0146] in which formula X is hydrogen or hydroxy; R¹ and R², which maybe the same or different, stand for hydrogen or a C₁-C₆ hydrocrabylradical; or R¹ and R², taken together with the carbon atom (starred informula I) bearing the group X, can form a C₃-C₈ carbocyclic ring; R³stands for hydrogen or a C₁-C₁₀ hydrocarbyl radial or for YR⁴, in whichY stands for the radicals —CO—, —CO—, —CO—S—, —CS—, —CS—O, —CS—S—,S—SO—OR—SO₂—, and R⁴ stands for hydrogen or a C₁-C₁₀ hydrocarbylradical; Qls a single bond or a C₁-C₈ hydrocarbylene diradical; R¹, R²,R³, and/or Q may be optionally substituted with one or more deuterium orfluorine atoms.

[0147] In particular a diastereoisomer of a compound mentioned above, inpure form; or a mixture of diastereoisomers of a compound mentionedabove.

[0148] Moreover, a compound according to the above mentionedspecifications, selected from the group consisting of

[0149]1(S),3(R)-Dihydroxy-20(R)-(1,5-dihydroxy-5-ethyl-2-heptyn-1-yl)-9,10-seco-pregna-5(Z),7(E),10(19)-triene;

[0150]1(S),3(R)-Dihydroxy-20(R)-(5-etyl-5-hydroxy-1-methoxy-2-heptyn-1-yl)-9,10-seco-pregna-5(Z),7(E),10(19)-triene;

[0151]1(S),3(R)-Dihydroxy-20(R)-(1-ethoxy-5-ehtyl-5-hydroxy-2-heptyn-1-yl)-9,10)-seco-pregna-5(Z),7(E),10(19)-triene;

[0152]1(S),3(R)-Dihydroxy-20(R)-(1-methoxy-4-hydroxy-4-ethyl-2-hexyn-1-yl)-9,10-seco-pregna-5(Z),7(E),10(19)-triene;

[0153]1(S),3(R)-Dihydroxy-20(R)-(1-ethoxy-4-hydroxy-4-ethyl-2-hexyn-1-yl)-9,10)-seco-pregna-5(Z),7(E),10(19)-triene;

[0154] isomer A.

[0155] WO 94/14766, which is hereby incorporated by reference, mentionsthe following vitamin D analogues:

[0156] A compound of the formula I

[0157] in which formula Y Is sulfur, S(O), or S(O)₂; R represents C₁-C₃alkyl; or

[0158] can form a C₃-C₈ carbocyclic ring; Q is a C₁-C₈ hydrocarbylenediradical; and prodrugs of I in which one or more of the hydroxy groupsare masked as groups which can be reconverted to hydroxy groups in vivo.

[0159] A compound of the formula I shown above in which Y Is sulfur andQ is C₂-C₄-alkylene.

[0160] Moreover a stereoisomer of a compound according to the abovementioned specifications, in pure form; or a mixture of suchstereoisomers.

[0161] Furthermore, a stereoisomer of a compound according to the abovementioned specifications having a saturated side chain with theR-configuration at C-20.

[0162] Furthermore, a compound according to the formula I which is

[0163] a)1(S),3(R)-Dihydroxy-20(R)-(4-ethyl-4-hydroxy-1-hexylthio)-9,10-seco-pregna-S(Z),7(E),10(19)-triene,

[0164] b)1(S),3(R)-Dihydroxy-20(R)-[5-methyl-5-hydroxy-1-hexylthio]-9,10-seco-pregna-S(Z),7(E),10(19)-triene,

[0165] c) 1(S),3(R)-Dihydroxy-20(R)-[3-(1-methyl-1-hydroxyethyl)benzylthio]-9,10-seco-pregna-5(Z),7(E),10(19)-triene,or

[0166] d)1(S),3(R)-Dihydroxy-20(R)-(3-methyl-3-hydroxy-1-butylthio)-9,10-seco-pregna-S(Z),7(E),10(19)-triene.

[0167] WO 93/0909, which is hereby incorporated by reference, mentionsthe following vitamin D analogues:

[0168] Compounds of general formulae (I) and (II)

[0169] (wherein Y represents an alkylene or alkenylene group containingup to 4 carbon atoms; R¹ and R², which may be the same or different,each represents a hydrogen atom or a lower alkyl or cycloalkyl group ortogether with the nitrogen atom to which they are attached form aheterocyclic group; and R¹ and R⁴, which may be the same or different,each represents a hydrogen atom or an O-protecting group).

[0170] Moreover compounds according to the above mentioned compoundswherein Y represents a group of formula

—(R^(A))_(m)—(R^(B))_(n)—

[0171] (wherein R^(A) is —CH═CH—, R^(B) is —CH₂, m is 0, 1 or 2 and n is0 or an integer such that 2 m+n=1, 2, 3 or 4.

[0172] Furthermore, compounds according to the above mentioned compoundswherein Y is a C₂-C₄, alkylene group.

[0173] Compounds according to the above mentioned compounds wherein atleast one of R¹ and R² is other than hydrogen.

[0174] Compounds according to the above mentioned compounds wherein R¹and R² are selected from hydrogen atoms, methyl and cyclopropyl groups,or R¹R²N— represents a piperidino group.

[0175] Compounds according to the above mentioned compounds wherein R³and R⁴ represent etherifying silyl groups.

[0176] Compounds according to the above mentioned compounds wherein R³and R⁴ are selected from hydrogen atoms and metabolically labileetherfying or esterifying groups.

[0177] Furthermore, the following compounds

[0178]1α,3β-dihydroxy-9,10-seco-25-azacholesta-5(Z),7,10(19)trien-24-one;

[0179]1α,3β-dihydroxy-23,23-bishomo-24-aza-9,10-secocholesta-5(Z),7,10(19)-trien-24-one;

[0180]1α,3β-dihydroxy-27-nor-9,10-secocholesta-5(Z),7,10(19),22,24-pentaene-26-carboxylicacid, 26-dimethyl amide;

[0181]N,N-pentamethylene-1α,3β-dihydroxy-9,10-secocholanamide-5(Z),7,10(19)-triene;

[0182]N-cyclopropyl-1α,3β-dihydroxy-9,10-secocholanamide-5(Z),7,10-(19)-triene;

[0183] 1α,3β-dihydroxy-9,10-secocholanamide-5(Z),7,10(19)-triene;

[0184]N,N-pentamethylene-1α,3β-dihydroxy-9,10-seco-20-epi-cholanamide-5(Z),7,10(19)triene;

[0185] and corresponding 5(E)-isomers thereof.

[0186] WO 94/26707, which is hereby incorporated by reference, mentionsthe following vitamin D analogues:

[0187] Compounds of general formula (I)

[0188] where R¹ and R², which may be the same or different, eachrepresents a hydrogen atom or an aliphatic, cycloaliphatic, araliphaticor aryl group or together with the nitrogen atom to which they areattached form a heterocyclic group; R³ represents a methyl group havingα- or β-configuration; X represents a valence bond or a C₁₋₂ alkylenegroup; Y represents —O—, —S—, —CH— or —NR— where R is a hydrogen atom oran organic group; Z represents a valence bond or a C₁₋₃ alkylene group;and A=represents a cyclohexylidiene moiety characteristic of the A-ringof a 1α-hydroxylated vitamin D or analogue thereof, with the provisothat when X-Y-Z-together represent an alkylene group containing up to 4carbon atoms, A=does not carry an exocyclic methylene group at the10-position.

[0189] Compounds of the general formula (I) as shown above whereinA=represents one of the groups

[0190] (where R⁴ and R⁵, which may be the same or different, eachrepresents a hydrogen atom or an O-protecting group).

[0191] Moreover compounds of the general formula (I) and thespecification mentioned above wherein R⁴ and R⁵ represent etherifyingsilyl groups.

[0192] Moreover compounds of the general formula (I) and thespecification mentioned above wherein R⁴ and R⁵ are selected fromhydrogen atoms and metabolically labile etherifying or esterif)ringgroups.

[0193] Furthermore compounds of the general formula (I) according to theabove specification wherein A=represents one of the groups

[0194] Compounds of the above general formula (I) wherein R¹ and R² areselected from hydrogen atoms, C₁₋₆ alkyl groups, C₃₋₈ cycloalkyl groups,C₆₋₁₂ aryl C₁₋₄ alkyl and optionally substituted C₆₋₁₂ carbocyclic arylgroups.

[0195] Compounds of the above-specked general formula (I) wherein R¹ andR² are selected from hydrogen atoms, C₁₋₆ alkyl groups and C₃₋₈cycloalkyl groups.

[0196] Compounds of the above-specified general formula (I) wherein R¹and R² are selected from hydrogen atoms, methyl, ethyl, and cyclopropylgroups.

[0197] Furthermore, the following compounds

[0198] 1α,3β-dihydroxy-20-epi-22-oxa-9,10-secochola-5(Z),7,10(19)trienicacid, piperidine amide;

[0199]1α,3β-dihydroxy-20-epi-23-homo-22-oxa-9,10-secochola-5(Z),7,10(19)-trienicadd, di-methylamine, cyclopropylamine and piperidine amides;

[0200] 1α,3β-dihydroxy-20-epi-22-oxa-9,10-secochola-5(Z),7,10(19-trienicacid, morpholine amide;

[0201]1α,3β-dihydroxy-20-epi-23-bis-homo-22-oxa-9,10-secochola-5(Z),7,10(19)-trienicacid, piperidine amide;

[0202]1α,3β-dihydroxy-20-epi-23-homo-23-oxa-9,10-secochola5(Z),7,10(19)-trienicadd, piperidine amide;

[0203]1α,3β-dihydroxy-23-homo-23-oxa-9,10-secochola-5(Z),7,10(19)-trienicacid, diethylamine, cyclopropylamine and piperidine amides and 20-epianalogues thereof;

[0204] 1α,3β-dihydroxy-23-homo-23-oxa-9,10-secochola-5(Z),7-dienic add,diethylamine, cyclo-propylamine and piperidine amides and 29-epianalogues thereof:

[0205]1α,3β-dihydroxy-23-homo-23-oxa-10-spirocyclopropyl-9,10-secochola-5(Z),7-dienicacid, diethylamine, cyclopropylamine and piperidine amides and 20-epianalogues thereof;

[0206] 1α,3β-dihydroxy-9,10-secochola-5(Z),7-dienic acid, piperidineamide;

[0207] 1α,3β-dihydroxy-10-spirocyclopropyl-9,10-secochola-5(Z),7-dienicacid, piperidine amide;

[0208] 1α,3β-dihydroxy-23-homo-9,10-secochola-5(Z),7-dienic acid,piperidine amide;

[0209]1α,3β-dihydroxy-23-homo-10-spirocyclopropyl-9,10-secochola-5(Z),7-dienicacid, piperidine amide;

[0210] 1α,3β-dihydroxy-23-homo-19-nor-9,10-secochola-5,7-dienic add,piperidine amide;

[0211] 1α,3β-dihydroxy-26-epi-23-homo-9,10-secochola-5(Z),7-dienic acid,piperidine amide;

[0212]1α,3β-dihydroxy-20-epi-23-homo-9,10-seco-23-thiachola-5(Z),7,10(19)-trienicacid, piperidine amide;

[0213]23-aza-1α,3β-dihydroxy-20-epi-23-bis-homo-9,10-secochola-5(Z),7,10(19)-trienicadd, piperidine amide;

[0214]23-aza-1α,3β-dihydroxy-20-epi-23-homo-9,10-secochola-5(Z),7,10(19)-trienicacid, piperidine amide;

[0215] 1α,3β-dihydroxy-20-epi-19-nor-9,10-secochola-5,7-dienic add,piperidine amide:

[0216] 1α,3β-dihydroxy-23-homo-19-nor-23-oxa-9,10-secochola-5,7-dienicacid, piperidine amide;

[0217] 23-aza-1α,3β-dihydroxy-9,10-secochola-5(Z),7,10(19)-trienic acid,piperidine amide;

[0218]22-aza-1α,3β-dihydroxy-20-epi-23-homo-9,10-secochola-5(Z),7,10(19)-trienicacid, piperidine amide;

[0219] 23-aza-1α,3β-dihydroxy-9,10-secochola-5(Z),7,10(19)-trienic add,diethyl amide and the 20-epi analogue thereof;

[0220] 1α,3β-dihydroxy-9,10-secochola-5(Z),7,10(19)-trienic acid,N-methyl-N-phenyl amide and the 20-epi analogue thereof; and

[0221] 1α,3β-dihydroxy-9,10-secochola-5(Z),7-dienic acid,N-methyl-N-phenyl amide and the 20-epi analogue thereof.

[0222] WO 95/03273, which is hereby incorporated by reference, mentionsthe following vitamin D analogues:

[0223] Compounds of general formula (I):

[0224] where R¹ represents a methyl group having α- or β-configuration;W represents a valence bond or a C₁₋₅ alkylene group; X represents azideor an optionally substituted triazole group; and A=represents acyclohexylidene moiety characteristic of the A-ring of a 1α-hydroxylatedvitamin D or analogue thereof.

[0225] Moreover compounds of the general formula (I) as shown abovehaving the general formula (II)

[0226] wherein R¹, W and A are as defined in claim 1; Y represents avalence bond or a lower alkylene group attached to the 4- or 5-positionof the triazole ring; and Z represents either (I) a group —CO.NR²R³ inwhich R² and R³ may be the same or different and are selected fromhydrogen atoms, aliphatic, cycloaliphatic, araliphatic and aryl groups,or R² and R³ together with the nitrogen atom to which they are attachedform a heterocyclic group; or (II) a group —(R⁴)(R⁵)—OH in which R⁴ andR⁵ may be the same or different and are selected from hydrogen atoms,aliphatic, cycloaliphatic, araliphatic and aryl groups, or R² and R³together with the carbon atom to which they are attached form a C₃₋₈carbocyclic ring.

[0227] Furthermore the compounds:

[0228]20α-(3-azidopropyl)-1α,3β-dihydroxy-9,10-secopregna-5(Z),7,10(19)-triene;

[0229] 20α-azido-1α,3β-dihydroxy-9,10-secopregna-5(Z),7,10(19)-triene;

[0230]1α,3β-dihydroxy-20α-[4-(2-hydroxyprop-2-yl)1,2,3-triazol-1-yl]-9,10-secopregna-5(Z),7,10(19)-triene;

[0231]1α,3β-dihydroxy-20α-[4-(3-hydroxypent-3-yl)-1,2,3-hiazol-1-yl]-9,10-secopregna-5(Z),7,10(19)-triene;

[0232]1α,3β-dihydroxy-20α-[4-(2-hydroxyprop-2-yl)-1,2,3-tdazol-1-ylmethyl]-9,10-secopregna-5(Z),7,10(19)-triene;

[0233]1α,3β-dihydroxy-20β-[4-(3-hydroxypent-3-yl)-1,2,3-triazol-1-ylmethyl]-9,10-secopregna-5(Z),7,10(19)-triene;

[0234]1α,3β-dihydroxy-20α-[4-(3-hydroxypent-3-yl)-1,2,3-triazol-1-ylmethyl]-9,10-secopregna-5(Z),7,10(19)-triene;

[0235]1α,3β-dihydroxy-20α-[4-(N,N-pentamethylenecarbamoyl)-1,2,3-triazol-1-ylmethyl]-9,10-secopregna-5(Z),7,10(19)-triene;

[0236]1α,3β-dihydroxy-20α-[4-(N,N-diethylcarbamoyl)-1,2,3-triazol-1-ylmethyl]-9,10-seco-pregna-5(Z),7,10(19)-triene;

[0237]1α,3β-dihydroxy-20α-[4-(N-cyclopropylcarbamoyl)-1,2,3-triazol-1-ylmethyl]-9,10-seco-pregna-5(Z),7,10(19)-triene;

[0238]1α,3β-dihydroxy-20α-[4-(N,N-3-oxapentamethylencarbamoyl)-1,2,3-triazol-1-ylmethyl]-9,10-secopregna-5(Z),7,10(19)-triene;

[0239]1α,3β-dihydroxy-20α-[4-(N,N-diisopropylcarbamoyl)-1,2,3-triazol-1-ylmethyl]-9,10-seco-preg5(Z),7,10(19)-triene;

[0240]1α,3β-dihydroxy-20β-[4-(N,N-pentamethylencarbamoyl)-1,2,3-triazol-1-ylmethyl]-9,10-secopregna-5(Z),7,10(19)-triene;

[0241]1α,3β-dihydroxy-20β-[4-(N,N-diethylcarbamoyl)-1,2,3-triazol-1-ylmethyl]-9,10-secopregna-5(Z),7,10(19)-triene;

[0242]1α,3β-dihydroxy-20β-[4-(N-cyclopropylcarbamoyl)-1,2,3-triazol-1-ylmethyl]-9,10-seco-pregna-5(Z),7,10(19)-triene;

[0243]1α,3β-dihydroxy-20β-[4-(N,N-3-oxapentamethylenecarbamoyl)-1,2,3-triazol-ylmethyl]9,10-secopregna-5(Z),7,10(19)-triene;

[0244]1α,3β-dihydroxy-20β-[4-(N,N-diisopropylcarbamoyl)-1,2,3-triazol-1-ylmethyl]-9,10-seco-pregna-5(Z),7,10(19)-triene;

[0245]1α,3β-dihydroxy-20α-{2-[4-(N,N-pentamethylenecarbamoyl)-1,2,3-triazol-1-yl]ethyl)-9,10-secopregna-5(Z),7,10(19)-triene;

[0246] 1α,3β-dihydroxy20α-(2-[4-(N,N-diethylearbamoyl)-1,2,3-triazol-1-yl]ethyl)-9,10-seco-pregna-5(Z),7,10(19)-triene;

[0247]1α,3β-dihydroxy-20α-(2-[4-(N-cyclopropylcarbamoyl)-1,2,3-triazol-1-yl]ethyl)-9,10-seco-pregna-5(Z),7,10(19)-triene;

[0248]1α,3β-dihydroxy-20α-{2-[4-(N,N-3-oxapentamethylenecarbamoyl)-1,2,3-triazol-1-yl]-ethyl)-9,10-secopregna-5(Z),7,10(19)triene;

[0249]1α,3β-dihydroxy-20α-(2-[4-(N,N-diisopropylcarbamoyl)-1,2,3-triazol-1-yl]ethyl)-9,20-seco-pregna-5(Z),7,10(19)-triene;

[0250]1α,3β-dihydroxy-20α-[4-(1-hydroxycyclohex-1-yl)-1,2,3-triazol-1-ylmethyl)-9,10-seco-pregna-5(Z),7,10(19)triene;

[0251]1α,3β-dihydroxy-20α-(2-[4-(3-hydroxypent-3-yl)-1,2,3-triazol-1-yl)ethyl)-9,10-secopregna-5(Z),7,10(19)-triene;

[0252]1α,3β-dihydroxy-20β{2-[4-(3-hydroxypent-3-yl)-1,2,3-triazol-1-yl]ethyl)-9,10-secopregna-5(Z),7,10(19)triene;

[0253]1α,3β-dihydroxy-20α-(3-[4-(3-hydroxypent-3-yl)-1,2,3-riazol-1-yl]propyl)9,10-secopregna-5(Z),7,10(19)-triene;

[0254]1α,3β-dihydroxy-20α-(3-[4-(3-methyl-3-hydroxybutyl)-1,2,3-triazol-1-yl)propyl)-9,10-seco-pregna-5(Z),7,10(19)-triene;

[0255]1α,3β-dihydroxy-20α-{3-[4-(2-methyl-2-hydroxypentyl)-1,2,3-triazol-1-yl)propyl]-9,10-seco-pregna-5(Z),7,10(19)-triene;

[0256]1α,3β-dihydroxy-20α-(3-[4-(4-ethyl-4-hydroxyhexyl]-1,2,3-triazol-1-yl]propyl)-9,10-seco-pregna5(Z),7,10(19)-triene;

[0257]1α,3β-dihydroxy-20α-{3-[4-(2-hydroxybut-2-yl)-1,2,3-triazol-1-yl]propyl)-9,10-secopregna-5(Z),7,10(19)triene;

[0258]1α,3β-dihydroxy-20α-{3-[4-(4-methyl-2-hydroxypent-2-yl)-1,2,3-triazol-1-yl)propyl)-9,10-secopregna-5(Z),7,10(19)-triene;

[0259]1α,3β-dihydroxy-20α-{3-[4-(2,4-dimethyl-3-hydroxypent-3-yl)-1,2,3-triazol-1-yl]propyl)-9,10-secopregna-5(Z),7,10(19)-triene;

[0260]1α,3β-dihydroxy-20α-[4-(2-ethyl-2-hydroxybutyl)-1,2,3-triazol-1-ylmethyl]-9,10-secopregna-5(Z),7,10(19)-triene;

[0261]1α,3β-dihydroxy-20α-[5-(2-ethyl-2-hydroxybutyl)-1,2,3-triazol-ylmethyl)-9,10-seco-pregna-5(Z),7,10(19)triene;

[0262]1α,3β-dihydroxy-20α-[4-(3-hydroxypent-3-yl)-1,2,3-triazol-1-ylmethyl]-9,10-secopregna-5(E),7-diene;

[0263]1α,3β-dihydroxy-20α-(3-hydroxypent-3-yl)-1,2,3-triazol-1-ylmethyl)-9,10-secopregna-5(Z),7-diene;

[0264]1α,3β-dihydroxy-20α-[4-(3-hydroxypent-3-yl)-1,2,3-triazol-1-ylmethyl]-10-spirocyclopropyl-9,10-secopregna-5(E),7-diene;

[0265]1α,3β-dihydroxy-20α-[4-(3-hydroxypent-3-yl)-1,2,3-triazol-1-ylmethyl)-10-spirocyclopropyl-9,10-secopregna-5(Z),7-diene;

[0266]1α,3β-dihydroxy-20α-[4-(3-hydroxypent-3-yl)-1,2,3-triazol-1-ylmethyl)-19-nor-9,10-seco-pregna-5,7-diene;

[0267]1α,3β-dihydroxy-20α-[4-(3-methyl-3-hydroxybutyl)-1,2,3-triazol-1-ylmethyl)-9,10-seco-pregna-5(Z),7,10(19)triene;

[0268]1α,3β-dihydroxy-20α-[4-(2-methyl-2-hydroxypentyl)-1,2,3-triazol-1-ylmethyl]-9,10-seco-pregna-5(Z),7,10(19)triene;

[0269]1α,3β-dihydroxy-20α-[4-(4-ethyl-4-hydroxyhexyl)-1,2,3-triazol-1-ylmethyl)-9,10-seco-pregna-5(Z),7,10(19)triene;

[0270]1α,3β-dihydroxy-20α-[4-(2-hydroxybut-2-yl)-1,2,3-triazol-1-ylmethyl]-9,10-secopregna-5(Z),7,10(19)triene;

[0271]1α,3β-dihydroxy-20α-[4-(4-methyl-2-hydroxypent-2-yl)-1,2,3-triazol-1-ylmethyl]-9,10-seco-pregna-5(Z),7,10(19)triene;

[0272]1α,3β-dihydroxy-20α-[4-(2,4-dimethyl-3-hydroxypent-3-yl)-1,2,3-triazol-1-ylmethyl]-9,10-secopregna-5(Z),7,10(19)-triene;and

[0273]1α,3β-dihydroxy-20α-[4-(2-hydroxypheneth-2-yl)-1,2,3-triazol-1-ylmethyl)-9,10-seco-pregna-5(Z),7,19(19)triene.

[0274] WO 95/02577, which is hereby incorporated by reference, mentionsthe following vitamin D analogues:

[0275] A compound of the formula

[0276] in which formula X is hydrogen or hydroxy; R¹ and R², which maybe the same or different, represent hydrogen or C₁-C₄ hydrocarbyl; or R¹and R², taken together with the carbon atom bearing the group X, canform a C₃-C₈ carbocyclic ring; Q is a single bond or a C₁-C4hydrocarbylene diradical, the expression hydrocarbyl radical(hydrocarbylene diradical) indicating the residue after removal of 1 (2)hydrogen atom(s) from a straight, branched or cyclic saturated orunsaturated hydrocarbon; R¹, R² and/or Q may be optionally substitutedwith one or more fluorine atoms; and prodrugs of I in which one or moreof the hydroxy groups are masked as groups which can be reconverted tohydroxy groups in vivo.

[0277] European patent application No. 0 205 025 A1, which is herebyincorporated by reference, mentions the following vitamin D analogues:

[0278] A compound of the formula (I)

[0279] wherein R₁ is a hydrogen atom, a hydroxyl group or a protectedhydroxyl group and R₂ is a hydrogen atom or a protecting group,

[0280] such as

[0281] 26,26,26,27,27,27-Hexafluoro-1α-hydroxyvitamin D₃.

[0282] 26,26,26,27,27,27-Hexaflourovitamin D₃.

[0283] The COX inhibitors according to the invention in addition to theASA include other NSAIDS known in the art.

EXAMPLE

[0284] The objective of the present example is to investigate thechemopreventive effect of a combination of acetylsalicylic acid,1α,25(OH)₂-vitamin D₃ and calcium on induced lung and urinary bladdertumorigenesis in A/J mice. Mice are chosen as the test model because oftheir suitability in this type of study. The A/J mouse induced with(B(a)P) and NNK is a scientifically recognized model of lungtumorigenesis (Prokopzyk et al., 2000, Pilegaard et al., 1997, Hecht etal., 1999).

[0285] Resumé:

[0286] At start of the experiment 60 female A/J mice are divided into 3groups each of 20 mice. Group A mice are fed normal diet (5000 ppmcalcium), Group B mice are fed a diet low in calcium (2500 ppm) andGroup C mice are fed a diet containing 300 ppm acetylsalicylic acid,0.02 ug/kg 1α,25(OH)₂-vitamin D₃ and 7500 ppm calcium. The three groupsare fed on the three different diets in the entire experimental period.

[0287] After two weeks of feeding on the respective diets, tumorigenesisis induced in all 60 A/J female mice by peroral dosing of the lungcarcinogens benzo(a)pyrene (B(a)P) and4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) once a week for 10consecutive weeks.

[0288] 2 weeks after the last treatment with carcinogens, the mice areremoved from the experiment. Lungs and bladders are prepared and thenumber of lung and bladder tumours are counted.

[0289] The results show that Group C, receiving 300 ppm acetylsalicylicacid, 0.02 ug/kg 1α,25(OH)₂-vitamin D₃ and 7500 ppm calcium, has ahighly significantly reduced incidence of lung tumors, when compared toGroup A, which is fed a standard diet (p<0.001). This finding applies toboth the number of tumour-bearing animals and tumour multiplicity.

[0290] The same effect is observed on urinary bladder tumors, althoughat a less significant level (p<0.025) which is probably due to the lowernumber of tumors found in the urinary bladders.

[0291] Surprisingly, it is also found that the diet containing 2500 ppmof calcium has a chemopreventive effect on lung tumors when compared tothe standard diet containing 5000 ppm of calcium (p=0.05). There is noapparent biologic rationale for this finding.

[0292] Materials and Methods

[0293] Inducers:

[0294] Benzo(a)pyrene (B(a)P), Aldrich Chem. Co., 3 μmol/mouse p.o.

[0295] 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 3μmol/mouse p.o.

[0296] Vehicle is cotton seed oil.

[0297] B(a)P and NNK are dissolved in cotton seed oil each at aconcentration of 6 μmol/ml. The mice are dosed 0.5 ml p.o.

[0298] Experimental System:

[0299] 60 female A/J mice are supplied by Taconic, N.Y., USA. At startof the acclimatization period the mice which have SPF status are 6 weeksof age.

[0300] The mice are caged in standard polypropylene cages(l×w×h=40×33×15 cm), 5 mice are caged together.

[0301] Bedding is Lignocel, produced by Hahn & Co., Faserstoffwerk,Bredenbeck-Kronsburg, Germany. Bedding is changed twice a week in alaminar flow unit. Cages are housed in an underpressured isolator withHEPA-filters (Class EU10, withholding 98.5% of all particles >0.3 μm.Temperature is 18° C.-22° C., and is controlled via the ambientventilation system in the laboratory. Light cycle is 12-hour dark and12-hour light (lights on 06.00).

[0302] Drinking water is UV sterilized. Water bottles are changed once aweek during acclimatization and experiment.

[0303] Diets for the experiment are specifically manufactured byAltromin Denmark, “Brogården”, Søbredden 27, 2820 Denmark.

[0304] Diet A is Altromin 1324 standard diet containing 5000 ppm Calcium

[0305] Diet B is Altromin 1324 standard diet containing 2500 ppm Calcium

[0306] Diet C is Altromin 1324 standard diet containing 300 ppmacetylsalicylic acid (Nycomed Denmark A/S), 0.02 ug/kg1α,25(OH)₂-vitamin D₃ (Leo Pharmaceuticals A/S, Denmark) and 7500 ppmcalcium.

[0307] Diet is in the form of pellets and is irradiated as a mean ofsterilization. Diet is administered ad libitum.

[0308] Animal Health and Welfare:

[0309] The mice have FELASA SPF-status and the housing and changingsystem is technically designed to preserve the SPF-status during thestudy. Mice are handled by educated personnel under veterinarysupervision. Daily records and decisions are made concerning animalwelfare.

[0310] Pre-Experimental Procedures:

[0311] Randomisation and Labelling:

[0312] On the day of arrival, the animals are randomly picked from thecrates and successively allocated to 12 cages each containing 5 mice.The mice are individually labelled by ear punctures.

[0313] Acclimatisation and Health Procedures:

[0314] Mice are imported to the laboratory at the start up of theexperimental procedure. No acclimatisation is needed since the mice arehoused under preexperimental conditions for the first 2 weeks.Suppliers' Health Monitoring Report regarding the mice is checked andthe mice are housed under conditions that preserve the SPF-status.

[0315] Experimental Procedures:

[0316] Upon arrival the mice are divided into 3 groups each of 20 mice.The groups are labelled A, B and C.

[0317] Group A is fed diet A.

[0318] Group B is fed diet B.

[0319] Group C is fed diet C.

[0320] The groups are fed these diets for the entire experimental period(16 weeks).

[0321] 2 weeks after the last treatment with carcinogens, the mice areremoved from the experiment. Lungs and bladders are prepared and thenumber of lung and bladder tumours are counted.

[0322] During the entire study period body weights, food consumption anddrinking water consumption is determined for each group once a week.

[0323] Terminal Procedures:

[0324] 2 weeks after the last treatment with carcinogens the mice areremoved from the experiment and examined for presence of lung andbladder tumours.

[0325] Mice are euthanized and gross pathology is performed.

[0326] After pathology, the lungs are removed and fixed inTelleysniczky's fixative (70% ethanol containing 5% glacial acetic acidand 5% of 4% neutral buffered formaldehyde).

[0327] After 7 days of fixation tumors, which appear as pearly noduleson the surface of the lungs and urinary bladders, are counted under adissecting microscope by two independent observers.

[0328] 10 random samples of nodules are taken from the lungs and urinarybladders for histological evaluation and confirmation of adenoma. Alltissues showing pathological changes are fixed in 4% bufferedformaldehyde, embedded in paraffin and stained with H/E.

[0329] Data Analysis

[0330] Tumor incidences in different treatment groups are compared bythe χ²-test. Food and water consumption in different treatment groupsare compared by paired F- and t-test.

[0331] Results

[0332] Lung Tumors:

[0333] Peroral dosing of B(a)P and (NNK) once a week for 10 consecutiveweeks result In a low number of lung tumors when the mice are terminated12 weeks after the first treatment. FIG. 2 shows the number of lungtumors in each group. The tumors are small, approximately 0.5-1 mm indiameter.

[0334] Statistical analysis, using the χ²-test for comparison of thetumor incidence between groups, shows that chemopreventive feeding withDiet C (300 ppm acetylsalicylic acid, 0.02 ug/kg 1α,25(OH)₂-vitamin D₃and 7500 ppm calcium significantly reduces tumor incidence when comparedto the Group A, which received standard diet containing 5000 ppm calcium(p<0.001).

[0335] The mice group B which is fed on Diet B containing 2500 ppmcalcium also has a lower tumor incidence than observed in Group A(p<0.05)

[0336] Urinary Bladder Tumors:

[0337] The number of urinary bladder tumors induced are lower than thenumber of lung tumors. The results are shown in FIG. 3.

[0338] Tumor incidence in urinary bladders also seem to be significantlyreduced when comparing Group C on chemopreventive diet to Group A onstandard diet (χ²-test, p<0.025). No significant reduction in tumorincidence is observed when low calcium Diet B is compared to Diet A.

[0339] Body Weights:

[0340] Body weights were measured weekly; starting in the week of firstinduction with carcinogen. No differences in body weights of the threegroups are observed and the growth curves appears very similar. Thus, ifbody weights are taken as a measure of the general health condition ofthe mice there appears to be no difference between the groups.

[0341] Food and Water Consumption:

[0342] Food consumption is also measured weekly. There appears to be atendency towards higher food consumption in Group C when compared toGroup A. Drinking water intake appears to be similar between group.

[0343] Clinical Observations:

[0344] A few mice died in each group during the experiment. The cause ofdeath was due to lesions caused by oral gavage.

[0345] From the results on body weights, food consumption and drinkingwater intake presented above it can be seen that the mice got into badcondition one week after dosing of carcinogens have started, but theygradually regained good condition during the rest of the study period.

[0346] During the study period a few incidences of mice with definedfirm swellings in the axillary regions of the front legs and in thedorsal region of the neck were observed. No differences were observedbetween groups. The results shown that carcinogenic treatment inducessubcutaneous transudates and cysts in the axillary regien of the frontlegs. Occationally hydrothorax is also observed.

CONCLUSION

[0347] Based on the results presented above it is concluded thatchemopreventive feeding on a diet composed of 300 ppm acetylsalicylicacid, 0.02 ug/kg 1α,25(OH)₂-vitamin D₃ and 7500 ppm calcium in a highlysignificant manner reduces the incidence of lung tumors in mice, whencompared to mice that were fed on a standard diet (p<0.001). Thisfinding applies to both number of tumour-bearing animals and tumourmultiplicity

[0348] The same effect was observed on urinary bladder tumors, althoughat a less significant level (p<0.025) which was probably due to the lownumber of tumors found in the urinary bladders.

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1. A method for the prevention of epithelial cancer or the initiationand/or progression of epithelial cancer in a human comprisingadministering to the human a combination dosage of a cyclooxygenase(COX) inhibitor, a vitamin D₃ including analogues and metabolitesthereof and calcium.
 2. A method according to claim 1 wherein the COXinhibitor is acetylsalicylic acid (ASA).
 3. A method for reducing theeffective dosage of ASA in a preventive treatment of epithelial cancerin a human by co-administration with a dosage of a vitamin D₃ includinganalogues and metabolites thereof and Ca, in the form of a combinationdosage.
 4. A method according to any of the preceding claims wherein thecombination dosage is administered regularly with an average dailydosage of ASA in the range of 50 mg to 500 mg.
 5. A method according toany of the preceding claims wherein the vitamin D₃ is 1,25dihydroxycholecalcipherol.
 6. A method according to any of the precedingclaims wherein the combination dosage comprises 1,25dihydroxycholecalcipherol in the range of 0.1 μg to 2 μg.
 7. A methodaccording to any of the preceding claims wherein the combination dosagecomprises calcium in the range of from 200 mg to 3000 mg.
 8. A methodaccording to any of the preceding claims wherein the human is selectedfrom the group being in risk of development of epithelial cancer due tobeing a first-degree relative to a patient with epithelial cancer,and/or carries the gene(s) for hereditary non-polyposis colorectalcancer (HNPCC).
 9. Use of a cyclooxygenase (COX) inhibitor, a vitamin D₃including analogues and metabolites thereof and calcium together with apharmaceutically acceptable carrier for the preparation of a medicamentfor preventing the initiation and/or progression of epithelial cancer ina human.
 10. Use according to claim 9 wherein the medicament is in theform of a combination dosage comprising the cyclooxygenase (COX)inhibitor, the vitamin D₃ including analogues and metabolites thereofand the calcium.
 11. Use according to claim 9 or 10 wherein the COXcyclooxygenase inhibitor is acetylsalicylic acid (ASA) in the range of50 mg to 500 mg.
 12. Use according to any of claims 9 to 11 wherein thecombination dosage comprises 1,25 dihydroxycholecalcipherol in the rangeof 0.1 μg to 2 μg.
 13. Use according to any of claims 9-12 wherein thecombination dosage comprises calcium in the range of 200 mg to 3000 mg.14. Use according to any of claims 10-13 wherein the combination dosagecomprises ASA, 1,25 DHC and Ca.
 15. A method according to any of claims1-8 or use according to any of claims 9-14 wherein the epithelial canceris selected from the group consisting of lung cancer, bladder cancer,prostate cancer and gynaecological cancer.